RESUMO
The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life1. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, which assemble together with the RuvA-Holliday junction complex to energize the strand-exchange reaction2. Despite its importance for chromosome maintenance, the structure and mechanism by which this complex facilitates branch migration are unknown. Here, using time-resolved cryo-electron microscopy, we obtained structures of the ATP-hydrolysing RuvAB complex in seven distinct conformational states, captured during assembly and processing of a Holliday junction. Five structures together resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange and context-specific conformational changes in RuvB. Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. Immobilization of the converter enables RuvB to convert the ATP-contained energy into a lever motion, which generates the pulling force driving the branch migration. We show that RuvB motors rotate together with the DNA substrate, which, together with a progressing nucleotide cycle, forms the mechanistic basis for DNA recombination by continuous branch migration. Together, our data decipher the molecular principles of homologous recombination by the RuvAB complex, elucidate discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors and provide a blueprint for the design of state-specific compounds targeting AAA+ motors.
Assuntos
ATPases Associadas a Diversas Atividades Celulares , Proteínas de Bactérias , DNA Helicases , DNA Cruciforme , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/ultraestrutura , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , DNA Helicases/química , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , DNA Cruciforme/química , DNA Cruciforme/metabolismo , DNA Cruciforme/ultraestrutura , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Recombinação Homóloga , Hidrólise , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Nucleotídeos , Conformação Proteica , RotaçãoRESUMO
After transcription termination, cellular RNA polymerases (RNAPs) are occasionally trapped on DNA, impounded in an undefined post-termination complex (PTC), limiting the free RNAP pool and subsequently leading to inefficient transcription. In Escherichia coli, a Swi2/Snf2 family of ATPase called RapA is known to be involved in countering such inefficiency through RNAP recycling; however, the precise mechanism of this recycling is unclear. To better understand its mechanism, here we determined the structures of two sets of E. coli RapA-RNAP complexes, along with the RNAP core enzyme and the elongation complex, using cryo-EM. These structures revealed the large conformational changes of RNAP and RapA upon their association that has been implicated in the hindrance of PTC formation. Our results along with DNA-binding assays reveal that although RapA binds RNAP away from the DNA-binding main channel, its binding can allosterically close the RNAP clamp, thereby preventing its nonspecific DNA binding and PTC formation. Taken together, we propose that RapA acts as a guardian of RNAP by which RapA prevents nonspecific DNA binding of RNAP without affecting the binding of promoter DNA recognition σ factor, thereby enhancing RNAP recycling.
Assuntos
Adenosina Trifosfatases/química , DNA Bacteriano/química , RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Complexos Multienzimáticos/química , Adenosina Trifosfatases/metabolismo , Microscopia Crioeletrônica , DNA Bacteriano/metabolismo , DNA Bacteriano/ultraestrutura , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/metabolismo , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestruturaRESUMO
DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Piridazinas/química , Monofosfato de Adenosina/química , Varredura Diferencial de Calorimetria , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Microscopia Crioeletrônica , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Endorribonucleases/química , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinética , Espectrometria de Massas , Complexos Multienzimáticos/ultraestrutura , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Piridazinas/farmacologia , Proteínas Recombinantes , Tetra-Hidroisoquinolinas/químicaRESUMO
In eukaryotes, a nascent peptide entering the endoplasmic reticulum (ER) is scanned by two Sec61 translocon-associated large membrane machines for protein N-glycosylation and protein O-mannosylation, respectively. While the structure of the eight-protein oligosaccharyltransferase complex has been determined recently, the structures of mannosyltransferases of the PMT family, which are an integral part of ER protein homeostasis, are still unknown. Here we report cryo-EM structures of the Saccharomyces cerevisiae Pmt1-Pmt2 complex bound to a donor and an acceptor peptide at 3.2-Å resolution, showing that each subunit contains 11 transmembrane helices and a lumenal ß-trefoil fold termed the MIR domain. The structures reveal the substrate recognition model and confirm an inverting mannosyl-transferring reaction mechanism by the enzyme complex. Furthermore, we found that the transmembrane domains of Pmt1 and Pmt2 share a structural fold with the catalytic subunits of oligosaccharyltransferases, confirming a previously proposed evolutionary relationship between protein O-mannosylation and protein N-glycosylation.
Assuntos
Manosiltransferases/ultraestrutura , Complexos Multienzimáticos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/enzimologia , Microscopia Crioeletrônica , Glicosilação , Humanos , Processamento de Imagem Assistida por Computador , Manose/metabolismo , Manosiltransferases/química , Manosiltransferases/genética , Manosiltransferases/metabolismo , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Especificidade por Substrato , Síndrome de Walker-Warburg/genéticaRESUMO
Oligosaccharyltransferase (OST) is an essential membrane protein complex in the endoplasmic reticulum, where it transfers an oligosaccharide from a dolichol-pyrophosphate-activated donor to glycosylation sites of secretory proteins. Here we describe the atomic structure of yeast OST determined by cryo-electron microscopy, revealing a conserved subunit arrangement. The active site of the catalytic STT3 subunit points away from the center of the complex, allowing unhindered access to substrates. The dolichol-pyrophosphate moiety binds to a lipid-exposed groove of STT3, whereas two noncatalytic subunits and an ordered N-glycan form a membrane-proximal pocket for the oligosaccharide. The acceptor polypeptide site faces an oxidoreductase domain in stand-alone OST complexes or is immediately adjacent to the translocon, suggesting how eukaryotic OSTs efficiently glycosylate a large number of polypeptides before their folding.
Assuntos
Hexosiltransferases/química , Proteínas de Membrana/química , Complexos Multienzimáticos/química , Proteínas de Saccharomyces cerevisiae/química , Domínio Catalítico , Sequência Conservada , Microscopia Crioeletrônica , Glicosilação , Hexosiltransferases/ultraestrutura , Proteínas de Membrana/ultraestrutura , Complexos Multienzimáticos/ultraestrutura , Oxirredução , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Especificidade por SubstratoRESUMO
Phosphatidylinositol 3-phosphate (PI3P) plays essential roles in vesicular trafficking, organelle biogenesis and autophagy. Two class III phosphatidylinositol 3-kinase (PI3KC3) complexes have been identified in mammals, the ATG14L complex (PI3KC3-C1) and the UVRAG complex (PI3KC3-C2). PI3KC3-C1 is crucial for autophagosome biogenesis, and PI3KC3-C2 is involved in various membrane trafficking events. Here we report the cryo-EM structures of human PI3KC3-C1 and PI3KC3-C2 at sub-nanometer resolution. The two structures share a common L-shaped overall architecture with distinct features. EM examination revealed that PI3KC3-C1 "stands up" on lipid monolayers, with the ATG14L BATs domain and the VPS34 C-terminal domain (CTD) directly contacting the membrane. Biochemical dissection indicated that the ATG14L BATs domain is responsible for membrane anchoring, whereas the CTD of VPS34 determines the orientation. Furthermore, PI3KC3-C2 binds much more weakly than PI3KC3-C1 to both PI-containing liposomes and purified endoplasmic reticulum (ER) vesicles, a property that is specifically determined by the ATG14L BATs domain. The in vivo ER localization analysis indicated that the BATs domain was required for ER localization of PI3KC3. We propose that the different lipid binding capacity is the key factor that differentiates the functions of PI3KC3-C1 and PI3KC3-C2 in autophagy.
Assuntos
Classe II de Fosfatidilinositol 3-Quinases/química , Complexos Multienzimáticos/química , Complexos Multienzimáticos/ultraestrutura , Microscopia Crioeletrônica , Humanos , Domínios Proteicos , Estrutura Quaternária de ProteínaRESUMO
FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is involved in the biochemical pathway for converting riboflavin into FAD. Human FADS exists in different isoforms. Two of these have been characterized and are localized in different subcellular compartments. hFADS2 containing 490 amino acids shows a two domain organization: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and a resembling molybdopterin-binding (MPTb) domain. By a multialignment of hFADS2 with other MPTb containing proteins of various organisms from bacteria to plants, the critical residues for hydrolytic function were identified. A homology model of the MPTb domain of hFADS2 was built, using as template the solved structure of a T. acidophilum enzyme. The capacity of hFADS2 to catalyse FAD hydrolysis was revealed. The recombinant hFADS2 was able to hydrolyse added FAD in a Co(2+) and mersalyl dependent reaction. The recombinant PAPS reductase domain is not able to perform the same function. The mutant C440A catalyses the same hydrolytic function of WT with no essential requirement for mersalyl, thus indicating the involvement of C440 in the control of hydrolysis switch. The enzyme C440A is also able to catalyse hydrolysis of FAD bound to the PAPS reductase domain, which is quantitatively converted into FMN.
Assuntos
Coenzimas/química , Coenzimas/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Hidrolases/química , Metaloproteínas/química , Metaloproteínas/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Pteridinas/química , Pteridinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Coenzimas/ultraestrutura , Simulação por Computador , Ativação Enzimática , Flavina-Adenina Dinucleotídeo/química , Humanos , Hidrolases/metabolismo , Metaloproteínas/ultraestrutura , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Cofatores de Molibdênio , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Nucleotidiltransferases/ultraestrutura , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The recycling of secretory granule membrane proteins that reach the plasma membrane following exocytosis is poorly understood. As a model, peptidylglycine alpha-amidating monooxygenase (PAM), a granule membrane protein that catalyzes a final step in peptide processing was examined. Ultrastructural analysis of antibody internalized by PAM and surface biotinylation showed efficient return of plasma membrane PAM to secretory granules. Electron microscopy revealed the rapid movement of PAM from early endosomes to the limiting membranes of multivesicular bodies and then into intralumenal vesicles. Wheat germ agglutinin and PAM antibody internalized simultaneously were largely segregated when they reached multivesicular bodies. Mutation of basally phosphorylated residues (Thr(946), Ser(949)) in the cytoplasmic domain of PAM to Asp (TS/DD) substantially slowed its entry into intralumenal vesicles. Mutation of the same sites to Ala (TS/AA) facilitated the entry of internalized PAM into intralumenal vesicles and its subsequent return to secretory granules. Entry of PAM into intralumenal vesicles is also associated with a juxtamembrane endoproteolytic cleavage that releases a 100-kDa soluble PAM fragment that can be returned to secretory granules. Controlled entry into the intralumenal vesicles of multivesicular bodies plays a key role in the recycling of secretory granule membrane proteins.
Assuntos
Proteínas de Membrana/metabolismo , Vesículas Secretórias/metabolismo , Animais , Biotinilação , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proteínas de Membrana/ultraestrutura , Camundongos , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/ultraestrutura , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Corpos Multivesiculares/metabolismo , Fosforilação , Neoplasias Hipofisárias/patologia , Treonina/metabolismoRESUMO
Replication of nuclear DNA in eukaryotes presents a tremendous challenge, not only due to the size and complexity of the genome, but also because of the time constraint imposed by a limited duration of S phase during which the entire genome has to be duplicated accurately and only once per cell division cycle. A challenge of this magnitude can only be met by the close coupling of DNA precursor synthesis to replication. Prokaryotic systems provide evidence for multienzyme and multiprotein complexes involved in DNA precursor synthesis and DNA replication. In addition, fractionation of nuclear proteins from proliferating mammalian cells shows co-sedimentation of enzymes involved in DNA replication with those required for synthesis of deoxynucleoside triphosphates (dNTPs). Such complexes can be isolated only from cells that are in S phase, but not from cells in G(0)/G(1) phases of cell cycle. The kinetics of deoxynucleotide metabolism supporting DNA replication in intact and permeabilized cells reveals close coupling and allosteric interaction between the enzymes of dNTP synthesis and DNA replication. These interactions contribute to channeling and compartmentation of deoxynucleotides in the microvicinity of DNA replication. A multienzyme and multiprotein megacomplex with these unique properties is called "replitase." In this article, we summarize some of the relevant evidence to date that supports the concept of replitase in mammalian cells, which originated from the observations in Dr. Pardee's laboratory. In addition, we show that androgen receptor (AR), which plays a critical role in proliferation and viability of prostate cancer cells, is associated with replitase, and that identification of constituents of replitase in androgen-dependent versus androgen-independent prostate cancer cells may provide insights into androgen-regulated events that control proliferation of prostate cancer cells and potentially offer an effective strategy for the treatment of prostate cancer.
Assuntos
Replicação do DNA , Complexos Multienzimáticos/metabolismo , Nucleosídeos/metabolismo , Animais , Ciclo Celular/fisiologia , Masculino , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/ultraestrutura , Nucleosídeos/química , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismoRESUMO
This communication illustrates how the electron microscope has contributed to biochemistry by revealing how multienzyme systems in mitochondria are structurally organized to secure high speed ATP synthesis and has extended physiology to the molecular level. Ribonucleoprotein complexes form a gel in the cytoplasm determining the conditions for translation... Photoreceptor stimulation involves two phases, trapping of light by a light reflecting cylinder formed by the outer segment disks and energy transduction by bleaching of photopigment molecules changing the charge of the outer segment disks driving the photoreceptor toward hyperpolarization. Revealing the synaptic connections between retinal neurons extends neurophysiology to the level of information processing by neural circuits, which are designed for high speed processing. Spatial brightness contrast enhancement is eliminated in connection with macular degeneration, which leads to partial blindness, revealing the importance of contrast enhancement for vision.
Assuntos
Artefatos , Membranas Intracelulares/ultraestrutura , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Complexos Multienzimáticos/ultraestrutura , Difosfato de Adenosina/fisiologia , Trifosfato de Adenosina/biossíntese , Animais , Respiração Celular/fisiologia , Citoplasma/química , Citoplasma/ultraestrutura , Técnica de Fratura por Congelamento/métodos , História do Século XX , Humanos , Membranas Intracelulares/enzimologia , Degeneração Macular/patologia , Degeneração Macular/fisiopatologia , Microscopia Eletrônica de Transmissão/história , Microscopia Eletrônica de Transmissão/métodos , Mitocôndrias/enzimologia , Complexos Multienzimáticos/fisiologia , Neurônios Aferentes/fisiologia , Neurônios Aferentes/ultraestrutura , Fosforilação , Células Fotorreceptoras/fisiologia , Células Fotorreceptoras/ultraestrutura , Ribonucleoproteínas/análise , Ribonucleoproteínas/ultraestrutura , Sinapses/fisiologia , Sinapses/ultraestruturaRESUMO
BACKGROUND: Prostasomes are small (40-500 nm), granule-like bodies, found in normal epithelial cells of the prostate and secreted into the prostate duct system. Also poorly differentiated prostate cancer cells are producing prostasomes, since we could isolate and purify prostasomes from vertebral metastases with biochemical methods. To find out whether these prostasomes are secreted into extracellular sites of the metastases, we used electron microscopy. METHODS: Small biopsies from vertebral metastases of prostate cancer, taken directly from the operating field at surgery, were immediately fixated, embedded in plastic and processed for electron microscopy. RESULTS: We found that prostasomes could be identified extracellularly in the interstitial tissues as well as in the cytoplasm of the metastatic cells. CONCLUSION: We conclude that prostasomes produced by the cells of vertebral metastases of prostate cancer are distributed both intracellularly and extracellularly in the interstitial spaces of the tissue. Thus, prostasomes of metastases could perhaps be exploited as targets for immunodiagnosis and/or immunotherapy.
Assuntos
Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Neoplasias da Próstata/secundário , Biópsia , Diferenciação Celular , Cisteína Endopeptidases/ultraestrutura , Citoplasma/ultraestrutura , Vesículas Citoplasmáticas/ultraestrutura , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Masculino , Microscopia Eletrônica , Complexos Multienzimáticos/ultraestrutura , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/ultraestrutura , Complexo de Endopeptidases do ProteassomaRESUMO
The SOR (sulphur oxygenase reductase) is the initial enzyme in the sulphur-oxidation pathway of Acidianus ambivalens. Expression of the sor gene in Escherichia coli resulted in active, soluble SOR and in inclusion bodies from which active SOR could be refolded as long as ferric ions were present in the refolding solution. Wild-type, recombinant and refolded SOR possessed indistinguishable properties. Conformational stability studies showed that the apparent unfolding free energy in water is approx. 5 kcal x mol(-1) (1 kcal=4.184 kJ), at pH 7. The analysis of the quaternary structures showed a ball-shaped assembly with a central hollow core probably consisting of 24 subunits in a 432 symmetry. The subunits form homodimers as the building blocks of the holoenzyme. Iron was found in the wild-type enzyme at a stoichiometry of one iron atom/subunit. EPR spectroscopy of the colourless SOR resulted in a single isotropic signal at g=4.3, characteristic of high-spin ferric iron. The signal disappeared upon reduction with dithionite or incubation with sulphur at elevated temperature. Thus both EPR and chemical analysis indicate the presence of a mononuclear iron centre, which has a reduction potential of -268 mV at pH 6.5. Protein database inspection identified four SOR protein homologues, but no other significant similarities. The spectroscopic data and the sequence comparison led to the proposal that the Acidianus ambivalens SOR typifies a new type of non-haem iron enzyme containing a mononuclear iron centre co-ordinated by carboxylate and/or histidine ligands.
Assuntos
Proteínas de Bactérias/química , Heme , Ferro/metabolismo , Oxirredutases/química , Acidianus/enzimologia , Sequência de Aminoácidos/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas Arqueais/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Dimerização , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica/genética , Microscopia Eletrônica/métodos , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Oxirredutases/ultraestrutura , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Conformação Proteica , Desnaturação Proteica/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/ultraestrutura , Especificidade por Substrato , Enxofre/metabolismoRESUMO
Ubiquitination is important for a broad array of cellular functions. Although reversal of this process, de-ubiquitination, most probably represents an important regulatory step contributing to cellular homeostasis, the specificity and properties of de-ubiquitination enzymes remain poorly understood. Here, we show that the Saccharomyces cerevisiae ubiquitin protease Ubp3 requires an additional protein, Bre5, to form an active de-ubiquitination complex that cleaves ubiquitin from specific substrates. In particular, this complex rescues Sec23p, a COPII subunit essential for the transport between the endoplasmic reticulum and the Golgi apparatus, from degradation by the proteasome. This probably contributes to maintaining and adapting a Sec23 expression level that is compatible with an efficient secretion pathway, and consequently with cell growth and viability.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas de Caenorhabditis elegans , Endopeptidases/deficiência , Galactosiltransferases/deficiência , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/ultraestrutura , Endopeptidases/genética , Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Proteínas Ativadoras de GTPase , Galactosiltransferases/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Microscopia Eletrônica , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Complexo de Endopeptidases do Proteassoma , Transporte Proteico/fisiologia , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina TiolesteraseRESUMO
Specific labelling with monoclonal antibodies reveals that in regulator-proteasome complexes the asymmetric 19S regulator (PA700) binds to one or both terminal alpha-disks of the cylinder-shaped 20S core proteasome in such a way that its reclining front part is positioned in the vicinity of proteasome subunit alpha6. The protruding rear part of the regulator appears to be situated distal to the sites occupied by the subunits alpha2 and alpha3, respectively. When viewed from beta1/beta1' to beta4/beta4' along the polar 2-fold axis of the 20S proteasome core, the rear part of each 19S regulator cap appears to protrude clockwise. Thus, a defined alignment of the 19S regulator with respect to the single polar 2-fold rotational axis of the 20S core proteasome is obtained.
Assuntos
Cisteína Endopeptidases/ultraestrutura , Complexos Multienzimáticos/ultraestrutura , Proteínas/ultraestrutura , Anticorpos Monoclonais , Cisteína Endopeptidases/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Microscopia Imunoeletrônica , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Conformação Proteica , Subunidades Proteicas , Proteínas/metabolismoRESUMO
The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that is activated by low cellular energy status and affects a switch away from energy-requiring processes and toward catabolism. While it is primarily regulated by AMP and ATP, high muscle glycogen has also been shown to repress its activation. Mutations in the gamma2 and gamma3 subunit isoforms lead to arrhythmias associated with abnormal glycogen storage in human heart and elevated glycogen in pig muscle, respectively. A putative glycogen binding domain (GBD) has now been identified in the beta subunits. Coexpression of truncated beta subunits lacking the GBD with alpha and gamma subunits yielded complexes that were active and normally regulated. However, coexpression of alpha and gamma with full-length beta caused accumulation of AMPK in large cytoplasmic inclusions that could be counterstained with anti-glycogen or anti-glycogen synthase antibodies. These inclusions were not affected by mutations that increased or abolished the kinase activity and were not observed by using truncated beta subunits lacking the GBD. Our results suggest that the GBD binds glycogen and can lead to abnormal glycogen-containing inclusions when the kinase is overexpressed. These may be related to the abnormal glycogen storage bodies seen in heart disease patients with gamma2 mutations.
Assuntos
Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Glicogênio/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Arritmias Cardíacas/enzimologia , Linhagem Celular Tumoral , Glicogênio Sintase/química , Glicogênio Sintase/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/ultraestrutura , Testes de Precipitina , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/ultraestrutura , Estrutura Terciária de Proteína , Subunidades Proteicas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de SequênciaRESUMO
The proteasome, a large non-lysosomal multi-subunit protease complex, is ubiquitous in eukaryotic cells. In protozoan parasites, the proteasome is involved in cell differentiation and replication, and could therefore be a promising therapeutic target. This article reviews the present knowledge of proteasomes in protozoan parasites of medical importance such as Giardia, Entamoeba, Leishmania, Trypanosoma, Plasmodium and Toxoplasma spp.
Assuntos
Cisteína Endopeptidases/fisiologia , Eucariotos/enzimologia , Complexos Multienzimáticos/fisiologia , Inibidores de Proteases/uso terapêutico , Infecções por Protozoários/parasitologia , Animais , Antiprotozoários , Cisteína Endopeptidases/química , Cisteína Endopeptidases/ultraestrutura , Entamoeba/enzimologia , Entamoeba/fisiologia , Eucariotos/fisiologia , Giardia/enzimologia , Giardia/fisiologia , Leishmania/enzimologia , Leishmania/fisiologia , Complexos Multienzimáticos/química , Complexos Multienzimáticos/ultraestrutura , Plasmodium/enzimologia , Plasmodium/fisiologia , Complexo de Endopeptidases do Proteassoma , Toxoplasma/enzimologia , Toxoplasma/fisiologia , Trypanosoma/enzimologia , Trypanosoma/fisiologiaRESUMO
Newly synthesized proteins that do not fold correctly in the ER are targeted for ER-associated protein degradation (ERAD) through distinct sorting mechanisms; soluble ERAD substrates require ER-Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER. Retained transmembrane proteins are often sequestered into specialized ER subdomains, but the relevance of such sequestration to proteasomal degradation has not been explored. We used the yeast Saccharomyces cerevisiae and a model ERAD substrate, the cystic fibrosis transmembrane conductance regulator (CFTR), to explore whether CFTR is sequestered before degradation, to identify the molecular machinery regulating sequestration, and to analyze the relationship between sequestration and degradation. We report that CFTR is sequestered into ER subdomains containing the chaperone Kar2p, and that sequestration and CFTR degradation are disrupted in sec12ts strain (mutant in guanine-nucleotide exchange factor for Sar1p), sec13ts strain (mutant in the Sec13p component of COPII), and sec23ts strain (mutant in the Sec23p component of COPII) grown at restrictive temperature. The function of the Sar1p/COPII machinery in CFTR sequestration and degradation is independent of its role in ER-Golgi traffic. We propose that Sar1p/COPII-mediated sorting of CFTR into ER subdomains is essential for its entry into the proteasomal degradation pathway. These findings reveal a new aspect of the degradative mechanism, and suggest functional crosstalk between the secretory and the degradative pathways.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Membrana Celular/metabolismo , Cisteína Endopeptidases/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Eucarióticas/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multienzimáticos/metabolismo , Transporte Proteico/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Compartimento Celular/fisiologia , Membrana Celular/ultraestrutura , Cisteína Endopeptidases/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Células Eucarióticas/ultraestrutura , Proteínas Fúngicas/metabolismo , Proteínas Ativadoras de GTPase , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Complexos Multienzimáticos/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares , Complexo de Endopeptidases do Proteassoma , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte VesicularRESUMO
We have identified proteins that are abundant in affinity-purified proteasomes, but absent from proteasomes as previously defined because elevated salt concentrations dissociate them during purification. The major components are a deubiquitinating enzyme (Ubp6), a ubiquitin-ligase (Hul5), and an uncharacterized protein (Ecm29). Ecm29 tethers the proteasome core particle to the regulatory particle. Proteasome binding activates Ubp6 300-fold and is mediated by the ubiquitin-like domain of Ubp6, which is required for function in vivo. Ubp6 recognizes the proteasome base and its subunit Rpn1, suggesting that proteasome binding positions Ubp6 proximally to the substrate translocation channel. ubp6Delta mutants exhibit accelerated turnover of ubiquitin, indicating that deubiquitination events catalyzed by Ubp6 prevent translocation of ubiquitin into the proteolytic core particle.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Canavanina/metabolismo , Cisteína Endopeptidases/isolamento & purificação , Cisteína Endopeptidases/ultraestrutura , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Ligases/genética , Ligases/isolamento & purificação , Ligases/metabolismo , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/ultraestrutura , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Sais/químicaRESUMO
It is generally accepted that the damage recognition complex of nucleotide excision repair in Escherichia coli consists of two UvrA and one UvrB molecule, and that in the preincision complex UvrB binds to the damage as a monomer. Using scanning force microscopy, we show here that the damage recognition complex consists of two UvrA and two UvrB subunits, with the DNA wrapped around one of the UvrB monomers. Upon binding the damage and release of the UvrA subunits, UvrB remains a dimer in the preincision complex. After association with the UvrC protein, one of the UvrB monomers is released. We propose a model in which the presence of two UvrB subunits ensures damage recognition in both DNA strands. Upon binding of the UvrA(2)B(2) complex to a putative damaged site, the DNA wraps around one of the UvrB monomers, which will subsequently probe one of the DNA strands for the presence of a lesion. When no damage is found, the DNA will wrap around the second UvrB subunit, which will check the other strand for aberrations.
Assuntos
Adenosina Trifosfatases/fisiologia , Dano ao DNA , DNA Helicases/fisiologia , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/fisiologia , Endodesoxirribonucleases/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Complexos Multienzimáticos/fisiologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/ultraestrutura , DNA Helicases/química , DNA Helicases/ultraestrutura , DNA Bacteriano/química , DNA Bacteriano/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/ultraestrutura , Dimerização , Endodesoxirribonucleases/química , Endodesoxirribonucleases/ultraestrutura , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Substâncias Macromoleculares , Microscopia de Força Atômica , Complexos Multienzimáticos/química , Complexos Multienzimáticos/ultraestrutura , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Subunidades Proteicas , Relação Estrutura-AtividadeRESUMO
Mutations in the photopigment rhodopsin are the major cause of autosomal dominant retinitis pigmentosa. The majority of mutations in rhodopsin lead to misfolding of the protein. Through the detailed examination of P23H and K296E mutant opsin processing in COS-7 cells, we have shown that the mutant protein does not accumulate in the Golgi, as previously thought, instead it forms aggregates that have many of the characteristic features of an aggresome. The aggregates form close to the centrosome and lead to the dispersal of the Golgi apparatus. Furthermore, these aggregates are ubiquitinated, recruit cellular chaperones and disrupt the intermediate filament network. Mutant opsin expression can disrupt the processing of normal opsin, as co-transfection revealed that the wild-type protein is recruited to mutant opsin aggregates. The degradation of mutant opsin is dependent on the proteasome machinery. Unlike the situation with DeltaF508-CFTR, proteasome inhibition does not lead to a marked increase in aggresome formation but increases the retention of the protein within the ER, suggesting that the proteasome is required for the efficient retrotranslocation of the mutant protein. Inhibition of N-linked glycosylation with tunicamycin leads to the selective retention of the mutant protein within the ER and increases the steady state level of mutant opsin. Glycosylation, however, has no influence on the biogenesis and targeting of wild-type opsin in cultured cells. This demonstrates that N-linked glycosylation is required for ER-associated degradation of the mutant protein but is not essential for the quality control of opsin folding. The addition of 9-cis-retinal to the media increased the amount of P23H, but not K296E, that was soluble and reached the plasma membrane. These data show that rhodopsin autosomal dominant retinitis pigmentosa is similar to many other neurodegenerative diseases in which the formation of intracellular protein aggregates is central to disease pathogenesis, and they suggest a mechanism for disease dominance.